Analogues of estradiol-17 beta (E2) have been evaluated for estrogen receptor (ER) binding affinity and mitogenic potential in the human breast cancer cell line MCF-7. These 42 compounds represent subtle modifications of the natural estrogen structure through the placement of hydroxyl, amino, nitro, or iodo groups around the ring system in addition to, or as replacement of, the 3- and 17 beta-hydroxyls of E2. The mitogenic activity of the analogues was found to be related to ER binding only to a limited extent. In order to elucidate structural features that are uniquely responsible for receptor binding affinity or mitogen potential of estrogens, the three-dimensional quantitative structure-activity (QSAR) method Comparative Molecular Field Analysis (CoMFA) was employed. Separate CoMFA models for receptor binding and cell growth stimulation were optimized through the use of various alignment rules and region step size. Whereas the CoMFA contour plots did outline the shared structural requirements for the two measured biological properties, specific topological features in this set of estrogens were delineated that distinguish mitogenic potential from ER binding ability. In particular, steric interference zones which affected growth extend in a band from above the A-ring to position 4 and below, whereas the ER binding steric interference zones are limited to isolated polyhedra in the 1, 2 and 4 positions and the alpha face of the B-ring. In addition, electronegative features located around the A-, B-, or C-rings contribute to receptor affinity. However, growth is dependent only on electronegative and electropositive properties near the 3-position. In a final QSAR model for the mitogenic response, the value of ER binding was included along with structural features as a descriptor in CoMFA. The resulting 3D-QSAR has the most predictive potential of the models in this study and can be considered a prototype model for the general evaluation of a steroidal estrogen's growth stimulating ability in MCF-7 cells. For example, the location of D-ring contours illustrate the model's preference for 17 beta-hydroxy steroids over the less mitogenic 17 alpha- and 16 alpha-hydroxy compounds. In addition, the enhanced mitogenic effect of steric bulk in the 11 alpha-position is also evident. The QSAR studies in this report illustrate the fact that while ER binding may be a required factor of the estrogen dependent growth response in MCF-7 cells, particular structural characteristics, in addition to those responsible for tight receptor binding, must be present to induce an optimal mitogenic response. Therefore, this report demonstrates that the CoMFA QSAR method can be utilized to characterize structural features of test compounds that account for different types of estrogenic responses.